PSMA Targeting Trispecific Proteins and Methods of Use

ABSTRACT

Provided herein are prostate specific membrane antigen (PSMA) targeting trispecific proteins comprising a domain binding to CD3, a half-life extension domain, and a domain binding to PSMA. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such PSMA targeting trispecific proteins. Also disclosed are methods of using the disclosed PSMA targeting trispecific proteins in the prevention, and/or treatment diseases, conditions and disorders.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 15/821,530, filed on Nov. 22, 2017, and claims thebenefit of U.S. Provisional Application Nos. 62/426,069 filed Nov. 23,2016, and 62/426,077 filed Nov. 23, 2016, which are incorporated byreference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 22, 2017, isnamed 47517-708_201_SL.txt and is 150,911 bytes in size.

BACKGROUND OF THE INVENTION

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged. One suchmethod is by inducing an immune response against the tumor, to makeimmune effector cells such as natural killer (NK) cells or cytotoxic Tlymphocytes (CTLs) attack and destroy tumor cells.

SUMMARY OF THE INVENTION

Provided herein are trispecific antigen-binding protein, pharmaceuticalcompositions thereof, as nucleic acids, recombinant expression vectorsand host cells for making such trispecific antigen-binding proteins, andmethods of use for the treatment of diseases, disorders, or conditions.In one aspect, described herein are prostate specific membrane antigen(PSMA) targeting trispecific proteins, wherein said proteins comprise(a) a first domain (A) which specifically binds to human CD3; (b) asecond domain (B) which is a half-life extension domain; and (c) a thirddomain (C) which specifically binds to PSMA, wherein the domains arelinked in the order H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH,H2N-(C)-(B)-(A)-COOH, or by linkers L1 and L2. In some embodiments, thefirst domain comprises a variable light chain and variable heavy chaineach of which is capable of specifically binding to human CD3. In someembodiments, the first domain comprises one or more sequences selectedfrom the group consisting of SEQ ID NO: 1-88. In some embodiments, thefirst domain is humanized or human. In some embodiments, the firstdomain has a KD binding of 150 nM or less to CD3 on CD3 expressingcells. In some embodiments, the second domain binds human serum albumin.In some embodiments, the second domain comprises a scFv, a variableheavy domain (VH), a variable light domain (VL), a peptide, a ligand, ora small molecule. In some embodiments, the second domain comprises oneor more sequences selected from the group consisting of SEQ ID NOs:89-112. In some embodiments, the third domain comprises a scFv, a VHdomain, a VL domain, a non-Ig domain, a ligand, a knottin, or a smallmolecule entity that specifically binds to PSMA. In some embodiments,the third domain comprises one or more sequences selected from the groupconsisting of SEQ ID NOs: 113-140.

In some embodiments, linkers L1 and L2 are each independently selectedfrom (GS)n (SEQ ID NO: 153), (GGS)n (SEQ ID NO: 154), (GGGS)n (SEQ IDNO: 155), (GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157), or(GGGGS)n (SEQ ID NO: 158), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. In some embodiments, linkers L1 and L2 are each independently(GGGGS)4 (SEQ ID NO: 159) or (GGGGS)3 (SEQ ID NO: 160). In someembodiments, the domains are linked in the order H2N-(A)-(C)-(B)-COOH.In some embodiments, the domains are linked in the orderH2N-(B)-(C)-(A)-COOH.

In some embodiments, the protein is less than about 80 kDa. In someembodiments, the protein is about 50 to about 75 kDa. In someembodiments, the protein is less than about 60 kDa. In some embodiments,the protein has an elimination half-time of at least about 50 hours. Insome embodiments, the protein has an elimination half-time of at leastabout 100 hours. In some embodiments, the protein has increased tissuepenetration as compared to an IgG to the same PSMA.

In some embodiments, the protein comprises a sequence selected from thegroup consisting of SEQ ID NO: 140-152.

In another aspect, provided herein are pharmaceutical compositioncomprising (i) the PSMA targeting trispecific protein according to anyone of the above embodiments and (ii) a pharmaceutically acceptablecarrier.

Also provided herein are methods of treating an individual in need oftreatment of cancer, the method comprising administration of aneffective amount of the pharmaceutical composition or PSMA targetingtrispecific proteins according to any of the above embodiments. In someembodiments, the cancer is prostate cancer or renal cancer.

One embodiment provides a PSMA targeting trispecific protein, whereinsaid protein comprises (a) a first domain (A) which specifically bindsto human CD3; (b) a second domain (B) which is a half-life extensiondomain; and (c) a third domain (C) which specifically binds to PSMA,wherein the second domain comprises one or more sequences selected fromthe group consisting of SEQ ID NOs: 113-140. In some embodiments,domains are linked in the order H₂N-(A)-(C)-(B)-COOH,H₂N-(B)-(A)-(C)-COOH, H₂N-(C)-(B)-(A)-COOH, or by linkers L1 and L2. Insome embodiments, the first domain comprises one or more sequencesselected from the group consisting of SEQ ID NO: 1-88. In someembodiments, the second domain comprises one or more sequences selectedfrom the group consisting of SEQ ID NO: 89-112.

One embodiment provides a PSMA targeting trispecific protein, whereinsaid protein comprises a sequence selected from the group consisting ofSEQ ID NO: 140-152. In some embodiments, said protein comprises asequence selected from the group consisting of SEQ ID NO: 150-152.

One embodiment provides a prostate specific membrane antigen (PSMA)targeting trispecific protein, wherein said protein comprises (a) afirst domain (A) which specifically binds to human CD3; (b) a seconddomain (B) which is a half-life extension domain; and (c) a third domain(C) which specifically binds to PSMA, wherein the domains are linked inthe order H₂N-(C)-(B)-(A)-COOH, or by linkers L1 and L2, and wherein thethird domain comprises one or more sequences selected from the groupconsisting of SEQ ID NO: 113-140.

One embodiment provides a PSMA targeting trispecific protein, whereinsaid protein comprises (a) a first domain (A) which specifically bindsto human CD3; (b) a second domain (B) which is a half-life extensiondomain; and (c) a third domain (C) which specifically binds to PSMA,wherein the domains are linked in the order H₂N-(C)-(B)-(A)-COOH, or bylinkers L1 and L2, and wherein the first domain comprises one or moresequences selected from the group consisting of SEQ ID NO: 1-88.

One embodiment provides a method of treating prostate cancer, the methodcomprising administration of an effective amount of a PSMA targetingtrispecific protein, wherein said protein comprises (a) a first domain(A) which specifically binds to human CD3; (b) a second domain (B) whichis a half-life extension domain; and (c) a third domain (C) whichspecifically binds to PSMA, wherein the domains are linked in the orderH₂N-(C)-(B)-(A)-COOH, or by linkers L1 and L2, and wherein the thirddomain comprises one or more sequences selected from the groupconsisting of SEQ ID NO: 113-140.

One embodiment provides a method of treating prostate cancer, the methodcomprising administration of an effective amount of a PSMA targetingtrispecific protein, wherein said protein comprises (a) a first domain(A) which specifically binds to human CD3; (b) a second domain (B) whichis a half-life extension domain; and (c) a third domain (C) whichspecifically binds to PSMA, wherein the domains are linked in the orderH₂N-(C)-(B)-(A)-COOH, or by linkers L1 and L2, and wherein the firstdomain comprises one or more sequences selected from the groupconsisting of SEQ ID NO: 1-88.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is schematic representation of an exemplary PMSA targetingtrispecific antigen-binding protein where the protein has an constantcore element comprising an anti-CDR3ε single chain variable fragment(scFv) and an anti-HSA variable heavy chain region; and a PMSA bindingdomain that can be a VH, scFv, a non-Ig binder, or ligand.

FIGS. 2A-B compare the ability of exemplary PSMA targeting trispecificproteins (PSMA targeting TriTAC molecules) with different affinities forCD3 to induce T cells to kill human prostate cancer cells. FIG. 2A showskilling by different PMSA targeting TriTAC molecules in prostate cancermodel LNCaP. FIG. 2B shows killing by different PMSA targeting TriTACmolecules in prostate cancer model 22Rv1. FIG. 2C shows EC50 values forPMSA targeting TriTAC in LNCaP and 22Rv1 prostate cancer models.

FIG. 3 shows the serum concentration of PSMA targeting TriTAC C236 inCynomolgus monkeys after i.v. administration (100 μg/kg) over threeweeks.

FIG. 4 shows the serum concentration of PSMA targeting TriTAC moleculeswith different CD3 affinities in Cynomolgus monkeys after i.v.administration (100 μg/kg) over three weeks.

FIGS. 5A-C show the ability of PSMA targeting TriTAC molecules withdifferent affinities for PSMA to induce T cells to kill the humanprostate cancer cell line LNCaP. FIG. 5A shows the experiment performedin the absence of human serum albumin with a PSMA targeting BiTE aspositive control. FIG. 5B shows the experiment performed in the presenceof human serum albumin with a PSMA targeting BiTE as positive control.FIG. 5C shows EC50 values for PMSA targeting TriTAC in the presence orabsence of HSA with a PSMA targeting BiTE as a positive control in LNCaPprostate cancer models.

FIG. 6 demonstrates the ability of PSMA targeting TriTAC molecules toinhibit tumor growth of human prostate cancer cells in a mouse xenograftexperiment.

FIGS. 7A-D illustrates the specificity of TriTAC molecules in cellkilling assays with target cell lines that do or do not express thetarget protein. FIG. 7A shows EGFR and PSMA expression in LNCaP,KMS12BM, and OVCAR8 cell lines. FIG. 7B shows killing of LNCaP tumorcells by PSMA, EGFR, and negative control TriTACs. FIG. 7C shows killingof KMS12BM tumor cells by PSMA, EGFR, and negative control TriTACs. FIG.7D shows killing of OVCAR8 cells by PSMA, EGFR, and negative controlTriTACs.

FIGS. 8A-D depict the impact of pre-incubation at 37° C. and freeze/thawcycles on TriTAC activity. FIG. 8A shows PSMA TriTAC C235 activity afterpre-incubation at 37° C. or freeze/thaw cycles. FIG. 8B shows PSMATriTAC C359 activity after pre-incubation at 37° C. or freeze/thawcycles. FIG. 8C shows PSMA TriTAC C360 activity after pre-incubation at37° C. or freeze/thaw cycles. FIG. 8D shows PSMA TriTAC C361 activityafter pre-incubation at 37° C. or freeze/thaw cycles.

FIGS. 9A-B depict the activity of a PSMA targeting TriTAC molecule ofthis disclosure in redirected T cell killing in T cell dependentcellular cytotoxicity assays (TDCC). FIG. 9A shows the impact of thePSMA targeting TriTAC molecule in redirecting cynomolgus peripheralblood mononuclear cells (PBMCs), from cynomolgus monkey donor G322, inkilling LNCaP cells. FIG. 9B shows the impact of the PSMA targetingTriTAC molecule in redirecting cynomolgus PBMCs, from cynomolgus monkeydonor D173, to kill MDAPCa2b cells.

FIG. 10 depicts the impact of a PSMA targeting TriTAC molecule of thisdisclosure on expression of T cell activation markers CD25 and CD69.

FIG. 11 depicts the ability of a PSMA targeting TriTAC molecule of thisdisclosure to stimulate T cell proliferation in the presence of PSMAexpressing target cells.

FIGS. 12A-B depict redirected T cell killing of LnCaP cells by PSMAtargeting TriTAC molecules. FIG. 12A shows redirected T cell killing ofLnCaP cells by PSMA PH1T TriTAC (SEQ ID No: 150) and PSMA PH1 TriTAC(SEQ ID NO: 151) molecules. FIG. 12B shows redirected T cell killing ofLnCaP cells by PSMA Z2 TriTAC (SEQ ID NO: 152).

DETAILED DESCRIPTION OF THE INVENTION

Described herein are trispecific proteins that target prostate specificmembrane antigen (PSMA), pharmaceutical compositions thereof, as well asnucleic acids, recombinant expression vectors and host cells for makingsuch proteins thereof. Also provided are methods of using the disclosedPSMA targeting trispecific proteins in the prevention, and/or treatmentof diseases, conditions and disorders. The PSMA targeting trispecificproteins are capable of specifically binding to PSMA as well as CD3 andhave a half-life extension domain, such as a domain binding to humanserum albumin (HSA). FIG. 1 depicts one non-limiting example of atrispecific antigen-binding protein.

In one aspect, the PSMA targeting trispecific proteins comprise a domain(A) which specifically binds to CD3, a domain (B) which specificallybinds to human serum albumin (HSA), and a domain (C) which specificallybinds to PSMA. The three domains in PSMA targeting trispecific proteinsare arranged in any order. Thus, it is contemplated that the domainorder of the PSMA targeting trispecific proteins are:

-   -   H₂N-(A)-(B)-(C)-COOH,    -   H₂N-(A)-(C)-(B)-COOH,    -   H₂N-(B)-(A)-(C)-COOH,    -   H₂N-(B)-(C)-(A)-COOH,    -   H₂N-(C)-(B)-(A)-COOH, or    -   H₂N-(C)-(A)-(B)-COOH.

In some embodiments, the PSMA targeting trispecific proteins have adomain order of H₂N-(A)-(B)-(C)-COOH. In some embodiments, the PSMAtargeting trispecific proteins have a domain order ofH₂N-(A)-(C)-(B)-COOH. In some embodiments, the PSMA targetingtrispecific proteins have a domain order of H₂N-(B)-(A)-(C)-COOH. Insome embodiments, the PSMA targeting trispecific proteins have a domainorder of H₂N-(B)-(C)-(A)-COOH. In some embodiments, the PSMA targetingtrispecific proteins have a domain order of H₂N-(C)-(B)-(A)-COOH. Insome embodiments, the PSMA targeting trispecific proteins have a domainorder of H₂N-(C)-(A)-(B)-COOH.

In some embodiments, the PSMA targeting trispecific proteins have theHSA binding domain as the middle domain, such that the domain order isH₂N-(A)-(B)-(C)-COOH or H₂N-(C)-(B)-(A)-COOH. It is contemplated that insuch embodiments where the HSA binding domain as the middle domain, theCD3 and PSMA binding domains are afforded additional flexibility to bindto their respective targets.

In some embodiments, the PSMA targeting trispecific proteins describedherein comprise a polypeptide having a sequence described in Table 10(SEQ ID NO: 140-152) and subsequences thereof. In some embodiments, thetrispecific antigen binding protein comprises a polypeptide having atleast 70%-95% or more homology to a sequence described in Table 10 (SEQID NO: 140-152). In some embodiments, the trispecific antigen bindingprotein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%,95%, or more homology to a sequence described in Table 10 (SEQ ID NO:140-152). In some embodiments, the trispecific antigen binding proteinhas a sequence comprising at least a portion of a sequence described inTable 10 (SEQ ID NO: 140-152). In some embodiments, the PSMA trispecificantigen-binding protein comprises a polypeptide comprising one or moreof the sequences described in Table 10 (SEQ ID NO: 140-152). In furtherembodiments, the PSMA trispecific antigen-binding protein comprises oneor more CDRs as described in the sequences in Table 10 (SEQ ID NO:140-152).

The PSMA targeting trispecific proteins described herein are designed toallow specific targeting of cells expressing PSMA by recruitingcytotoxic T cells. This improves efficacy compared to ADCC (antibodydependent cell-mediated cytotoxicity), which is using full lengthantibodies directed to a sole antigen and is not capable of directlyrecruiting cytotoxic T cells. In contrast, by engaging CD3 moleculesexpressed specifically on these cells, the PSMA targeting trispecificproteins can crosslink cytotoxic T cells with cells expressing PSMA in ahighly specific fashion, thereby directing the cytotoxic potential ofthe T cell towards the target cell. The PSMA targeting trispecificproteins described herein engage cytotoxic T cells via binding to thesurface-expressed CD3 proteins, which form part of the TCR. Simultaneousbinding of several PSMA trispecific antigen-binding protein to CD3 andto PSMA expressed on the surface of particular cells causes T cellactivation and mediates the subsequent lysis of the particular PSMAexpressing cell. Thus, PSMA targeting trispecific proteins arecontemplated to display strong, specific and efficient target cellkilling. In some embodiments, the PSMA targeting trispecific proteinsdescribed herein stimulate target cell killing by cytotoxic T cells toeliminate pathogenic cells (e.g., tumor cells expressing PSMA). In someof such embodiments, cells are eliminated selectively, thereby reducingthe potential for toxic side effects.

The PSMA targeting trispecific proteins described herein confer furthertherapeutic advantages over traditional monoclonal antibodies and othersmaller bispecific molecules. Generally, the effectiveness ofrecombinant protein pharmaceuticals depends heavily on the intrinsicpharmacokinetics of the protein itself. One such benefit here is thatthe PSMA targeting trispecific proteins described herein have extendedpharmacokinetic elimination half-time due to having a half-lifeextension domain such as a domain specific to HSA. In this respect, thePSMA targeting trispecific proteins described herein have an extendedserum elimination half-time of about two, three, about five, aboutseven, about 10, about 12, or about 14 days in some embodiments. Thiscontrasts to other binding proteins such as BiTE or DART molecules whichhave relatively much shorter elimination half-times. For example, theBiTE CD19×CD3 bispecific scFv-scFv fusion molecule requires continuousintravenous infusion (i.v.) drug delivery due to its short eliminationhalf-time. The longer intrinsic half-times of the PSMA targetingtrispecific proteins solve this issue thereby allowing for increasedtherapeutic potential such as low-dose pharmaceutical formulations,decreased periodic administration and/or novel pharmaceuticalcompositions.

The PSMA targeting trispecific proteins described herein also have anoptimal size for enhanced tissue penetration and tissue distribution.Larger sizes limit or prevent penetration or distribution of the proteinin the target tissues. The PSMA targeting trispecific proteins describedherein avoid this by having a small size that allows enhanced tissuepenetration and distribution. Accordingly, the PSMA targetingtrispecific proteins described herein, in some embodiments have a sizeof about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kDto about 70 kD, or about 50 kD to about 65 kD. Thus, the size of thePSMA targeting trispecific proteins is advantageous over IgG antibodieswhich are about 150 kD and the BiTE and DART diabody molecules which areabout 55 kD but are not half-life extended and therefore cleared quicklythrough the kidney.

In further embodiments, the PSMA targeting trispecific proteinsdescribed herein have an optimal size for enhanced tissue penetrationand distribution. In these embodiments, the PSMA targeting trispecificproteins are constructed to be as small as possible, while retainingspecificity toward its targets. Accordingly, in these embodiments, thePSMA targeting trispecific proteins described herein have a size ofabout 20 kD to about 40 kD or about 25 kD to about 35 kD to about 40 kD,to about 45 kD, to about 50 kD, to about 55 kD, to about 60 kD, to about65 kD. In some embodiments, the PSMA targeting trispecific proteinsdescribed herein have a size of about 50 kD, 49, kD, 48 kD, 47 kD, 46kD, 45 kD, 44 kD, 43 kD, 42 kD, 41 kD, 40 kD, about 39 kD, about 38 kD,about 37 kD, about 36 kD, about 35 kD, about 34 kD, about 33 kD, about32 kD, about 31 kD, about 30 kD, about 29 kD, about 28 kD, about 27 kD,about 26 kD, about 25 kD, about 24 kD, about 23 kD, about 22 kD, about21 kD, or about 20 kD. An exemplary approach to the small size isthrough the use of single domain antibody (sdAb) fragments for each ofthe domains. For example, a particular PSMA trispecific antigen-bindingprotein has an anti-CD3 sdAb, anti-HSA sdAb and an sdAb for PSMA. Thisreduces the size of the exemplary PSMA trispecific antigen-bindingprotein to under 40 kD. Thus in some embodiments, the domains of thePSMA targeting trispecific proteins are all single domain antibody(sdAb) fragments. In other embodiments, the PSMA targeting trispecificproteins described herein comprise small molecule entity (SME) bindersfor HSA and/or the PSMA. SME binders are small molecules averaging about500 to 2000 Da in size and are attached to the PSMA targetingtrispecific proteins by known methods, such as sortase ligation orconjugation. In these instances, one of the domains of PSMA trispecificantigen-binding protein is a sortase recognition sequence, e.g., LPETG(SEQ ID NO: 57). To attach a SME binder to PSMA trispecificantigen-binding protein with a sortase recognition sequence, the proteinis incubated with a sortase and a SME binder whereby the sortaseattaches the SME binder to the recognition sequence. Known SME bindersinclude MIP-1072 and MIP-1095 which bind to prostate-specific membraneantigen (PSMA). In yet other embodiments, the domain which binds to PSMAof PSMA targeting trispecific proteins described herein comprise aknottin peptide for binding PSMA. Knottins are disulfide-stabilizedpeptides with a cysteine knot scaffold and have average sizes about 3.5kD. Knottins have been contemplated for binding to certain tumormolecules such as PSMA. In further embodiments, domain which binds toPSMA of PSMA targeting trispecific proteins described herein comprise anatural PSMA ligand.

Another feature of the PSMA targeting trispecific proteins describedherein is that they are of a single-polypeptide design with flexiblelinkage of their domains. This allows for facile production andmanufacturing of the PSMA targeting trispecific proteins as they can beencoded by single cDNA molecule to be easily incorporated into a vector.Further, because the PSMA targeting trispecific proteins describedherein are a monomeric single polypeptide chain, there are no chainpairing issues or a requirement for dimerization. It is contemplatedthat the PSMA targeting trispecific proteins described herein have areduced tendency to aggregate unlike other reported molecules such asbispecific proteins with Fc-gamma immunoglobulin domains.

In the PSMA targeting trispecific proteins described herein, the domainsare linked by internal linkers L1 and L2, where L1 links the first andsecond domain of the PSMA targeting trispecific proteins and L2 linksthe second and third domains of the PSMA targeting trispecific proteins.Linkers L1 and L2 have an optimized length and/or amino acidcomposition. In some embodiments, linkers L1 and L2 are the same lengthand amino acid composition. In other embodiments, L1 and L2 aredifferent. In certain embodiments, internal linkers L1 and/or L2 are“short”, i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the internal linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the internal linker is a peptide bond. In certainembodiments, internal linkers L1 and/or L2 are “long”, i.e., consist of15, 20 or 25 amino acid residues. In some embodiments, these internallinkers consist of about 3 to about 15, for example 8, 9 or 10contiguous amino acid residues. Regarding the amino acid composition ofthe internal linkers L1 and L2, peptides are selected with propertiesthat confer flexibility to the PSMA targeting trispecific proteins, donot interfere with the binding domains as well as resist cleavage fromproteases. For example, glycine and serine residues generally provideprotease resistance. Examples of internal linkers suitable for linkingthe domains in the PSMA targeting trispecific proteins include but arenot limited to (GS)n (SEQ ID NO: 153), (GGS)_(n) (SEQ ID NO: 154),(GGGS)_(n) (SEQ ID NO: 155), (GGSG)_(n) (SEQ ID NO: 156), (GGSGG)_(n)(SEQ ID NO: 157), or (GGGGS)_(n) (SEQ ID NO: 158), wherein n is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker L1 and/or L2is (GGGGS)₄ (SEQ ID NO: 159) or (GGGGS)₃ (SEQ ID NO: 160).

CD3 Binding Domain

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, MEW) by the TCR. As part of the TCR, CD3 isa protein complex that includes a CD3γ (gamma) chain, a CD3δ (delta)chain, and two CD3ε (epsilon) chains which are present on the cellsurface. CD3 associates with the α (alpha) and β (beta) chains of theTCR as well as CD3ζ (zeta) altogether to comprise the complete TCR.Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodiesleads to T cell activation similar to the engagement of the T cellreceptor but independent of its clone-typical specificity.

In one aspect, the PSMA targeting trispecific proteins described hereincomprise a domain which specifically binds to CD3. In one aspect, thePSMA targeting trispecific proteins described herein comprise a domainwhich specifically binds to human CD3. In some embodiments, the PSMAtargeting trispecific proteins described herein comprise a domain whichspecifically binds to CD3γ. In some embodiments, the PSMA targetingtrispecific proteins described herein comprise a domain whichspecifically binds to CD3δ. In some embodiments, the PSMA targetingtrispecific proteins described herein comprise a domain whichspecifically binds to CD3ε.

In further embodiments, the PSMA targeting trispecific proteinsdescribed herein comprise a domain which specifically binds to the TCR.In certain instances, the PSMA targeting trispecific proteins describedherein comprise a domain which specifically binds the a chain of theTCR. In certain instances, the PSMA targeting trispecific proteinsdescribed herein comprise a domain which specifically binds the β chainof the TCR.

In certain embodiments, the CD3 binding domain of the PSMA targetingtrispecific proteins described herein exhibit not only potent CD3binding affinities with human CD3, but show also excellentcrossreactivity with the respective cynomolgus monkey CD3 proteins. Insome instances, the CD3 binding domain of the PSMA targeting trispecificproteins are cross-reactive with CD3 from cynomolgus monkey. In certaininstances, human:cynomolgous K_(D) ratios for CD3 are between 5 and 0.2.

In some embodiments, the CD3 binding domain of the PSMA trispecificantigen-binding protein can be any domain that binds to CD3 includingbut not limited to domains from a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody. In some instances, it is beneficial for the CD3 binding domainto be derived from the same species in which the PSMA trispecificantigen-binding protein will ultimately be used in. For example, for usein humans, it may be beneficial for the CD3 binding domain of the PSMAtrispecific antigen-binding protein to comprise human or humanizedresidues from the antigen binding domain of an antibody or antibodyfragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-CD3 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a humanized or human anti-CD3 binding domain describedherein, and/or one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a humanized or human anti-CD3 binding domain describedherein, e.g., a humanized or human anti-CD3 binding domain comprisingone or more, e.g., all three, LC CDRs and one or more, e.g., all three,HC CDRs.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human light chain variable region specific toCD3 where the light chain variable region specific to CD3 compriseshuman or non-human light chain CDRs in a human light chain frameworkregion. In certain instances, the light chain framework region is a λ(lamda) light chain framework. In other instances, the light chainframework region is a κ (kappa) light chain framework.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human heavy chain variable region specific toCD3 where the heavy chain variable region specific to CD3 compriseshuman or non-human heavy chain CDRs in a human heavy chain frameworkregion.

In certain instances, the complementary determining regions of the heavychain and/or the light chain are derived from known anti-CD3 antibodies,such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the anti-CD3 binding domain is a single chainvariable fragment (scFv) comprising a light chain and a heavy chain ofan amino acid sequence provided herein. As used herein, “single chainvariable fragment” or “scFv” refers to an antibody fragment comprising avariable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlepolypeptide chain, and wherein the scFv retains the specificity of theintact antibody from which it is derived. In an embodiment, the anti-CD3binding domain comprises: a light chain variable region comprising anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions) of an amino acid sequence of a light chainvariable region provided herein, or a sequence with 95-99% identity withan amino acid sequence provided herein; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a heavychain variable region provided herein, or a sequence with 95-99%identity to an amino acid sequence provided herein. In one embodiment,the humanized or human anti-CD3 binding domain is a scFv, and a lightchain variable region comprising an amino acid sequence describedherein, is attached to a heavy chain variable region comprising an aminoacid sequence described herein, via a scFv linker. The light chainvariable region and heavy chain variable region of a scFv can be, e.g.,in any of the following orientations: light chain variable region-scFvlinker-heavy chain variable region or heavy chain variable region-scFvlinker-light chain variable region.

In some instances, scFvs which bind to CD3 are prepared according toknown methods. For example, scFv molecules can be produced by linking VHand VL regions together using flexible polypeptide linkers. The scFvmolecules comprise a scFv linker (e.g., a Ser-Gly linker) with anoptimized length and/or amino acid composition. Accordingly, in someembodiments, the length of the scFv linker is such that the VH or VLdomain can associate intermolecularly with the other variable domain toform the CD3 binding site. In certain embodiments, such scFv linkers are“short”, i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the scFv linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the scFv linker is a peptide bond. In some embodiments,these scFv linkers consist of about 3 to about 15, for example 8, 9 or10 contiguous amino acid residues. Regarding the amino acid compositionof the scFv linkers, peptides are selected that confer flexibility, donot interfere with the variable domains as well as allow inter-chainfolding to bring the two variable domains together to form a functionalCD3 binding site. For example, scFv linkers comprising glycine andserine residues generally provide protease resistance. In someembodiments, linkers in a scFv comprise glycine and serine residues. Theamino acid sequence of the scFv linkers can be optimized, for example,by phage-display methods to improve the CD3 binding and production yieldof the scFv. Examples of peptide scFv linkers suitable for linking avariable light chain domain and a variable heavy chain domain in a scFvinclude but are not limited to (GS)_(n) (SEQ ID NO: 153), (GGS)_(n) (SEQID NO: 154), (GGGS)_(n) (SEQ ID NO: 155), (GGSG)_(n) (SEQ ID NO: 156),(GGSGG)_(n) (SEQ ID NO: 157), or (GGGGS)_(n) (SEQ ID NO: 158), wherein nis 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linkercan be (GGGGS)₄ (SEQ ID NO: 159) or (GGGGS)₃ (SEQ ID NO: 160). Variationin the linker length may retain or enhance activity, giving rise tosuperior efficacy in activity studies.

In some embodiments, CD3 binding domain of PSMA trispecificantigen-binding protein has an affinity to CD3 on CD3 expressing cellswith a K_(D) of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nMor less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, theCD3 binding domain of PSMA trispecific antigen-binding protein has anaffinity to CD3ε, γ, or δ with a K_(D) of 1000 nM or less, 500 nM orless, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM orless. In further embodiments, CD3 binding domain of PSMA trispecificantigen-binding protein has low affinity to CD3, i.e., about 100 nM orgreater.

The affinity to bind to CD3 can be determined, for example, by theability of the PSMA trispecific antigen-binding protein itself or itsCD3 binding domain to bind to CD3 coated on an assay plate; displayed ona microbial cell surface; in solution; etc. The binding activity of thePSMA trispecific antigen-binding protein itself or its CD3 bindingdomain of the present disclosure to CD3 can be assayed by immobilizingthe ligand (e.g., CD3) or the PSMA trispecific antigen-binding proteinitself or its CD3 binding domain, to a bead, substrate, cell, etc.Agents can be added in an appropriate buffer and the binding partnersincubated for a period of time at a given temperature. After washes toremove unbound material, the bound protein can be released with, forexample, SDS, buffers with a high pH, and the like and analyzed, forexample, by Surface Plasmon Resonance (SPR).

In some embodiments, CD3 binding domains described herein comprise apolypeptide having a sequence described in Table 7 (SEQ ID NO: 1-88) andsubsequences thereof. In some embodiments, the CD3 binding domaincomprises a polypeptide having at least 70%-95% or more homology to asequence described in Table 7 (SEQ ID NO: 1-88). In some embodiments,the CD3 binding domain comprises a polypeptide having at least 70%, 75%,80%, 85%, 90%, 95%, or more homology to a sequence described in Table 7(SEQ ID NO: 1-88). In some embodiments, the CD3 binding domain has asequence comprising at least a portion of a sequence described in Table7 (SEQ ID NO: 1-88). In some embodiments, the CD3 binding domaincomprises a polypeptide comprising one or more of the sequencesdescribed in Table 7 (SEQ ID NO: 1-88).

In certain embodiments, CD3 binding domain comprises an scFv with aheavy chain CDR1 comprising SEQ ID NO: 16, and 22-33. In certainembodiments, CD3 binding domain comprises an scFv with a heavy chainCDR2 comprising SEQ ID NO: 17, and 34-43. In certain embodiments, CD3binding domain comprises an scFv with a heavy chain CDR3 comprising SEQID NO: 18, and 44-53. In certain embodiments, CD3 binding domaincomprises an scFv with a light chain CDR1 comprising SEQ ID NO: 19, and54-66. In certain embodiments, CD3 binding domain comprises an scFv witha light chain CDR2 comprising SEQ ID NO: 20, and 67-79. In certainembodiments, CD3 binding domain comprises an scFv with a light chainCDR3 comprising SEQ ID NO: 21, and 80-86.

Half-Life Extension Domain

Contemplated herein are domains which extend the half-life of anantigen-binding domain. Such domains are contemplated to include but arenot limited to HSA binding domains, Fc domains, small molecules, andother half-life extension domains known in the art.

Human serum albumin (HSA) (molecular mass ˜67 kDa) is the most abundantprotein in plasma, present at about 50 mg/ml (600 μM), and has ahalf-life of around 20 days in humans. HSA serves to maintain plasma pH,contributes to colloidal blood pressure, functions as carrier of manymetabolites and fatty acids, and serves as a major drug transportprotein in plasma.

Noncovalent association with albumin extends the elimination half-timeof short lived proteins. For example, a recombinant fusion of an albuminbinding domain to a Fab fragment resulted in an in vivo clearance of 25-and 58-fold and a half-life extension of 26- and 37-fold whenadministered intravenously to mice and rabbits respectively as comparedto the administration of the Fab fragment alone. In another example,when insulin is acylated with fatty acids to promote association withalbumin, a protracted effect was observed when injected subcutaneouslyin rabbits or pigs. Together, these studies demonstrate a linkagebetween albumin binding and prolonged action.

In one aspect, the PSMA targeting trispecific proteins described hereincomprise a half-life extension domain, for example a domain whichspecifically binds to HSA. In some embodiments, the HSA binding domainof PSMA trispecific antigen-binding protein can be any domain that bindsto HSA including but not limited to domains from a monoclonal antibody,a polyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the HSA binding domain is asingle chain variable fragments (scFv), single-domain antibody such as aheavy chain variable domain (VH), a light chain variable domain (VL) anda variable domain (VHH) of camelid derived single domain antibody,peptide, ligand or small molecule entity specific for HSA. In certainembodiments, the HSA binding domain is a single-domain antibody. Inother embodiments, the HSA binding domain is a peptide. In furtherembodiments, the HSA binding domain is a small molecule. It iscontemplated that the HSA binding domain of PSMA trispecificantigen-binding protein is fairly small and no more than 25 kD, no morethan 20 kD, no more than 15 kD, or no more than 10 kD in someembodiments. In certain instances, the HSA binding is 5 kD or less if itis a peptide or small molecule entity.

The half-life extension domain of PSMA trispecific antigen-bindingprotein provides for altered pharmacodynamics and pharmacokinetics ofthe PSMA trispecific antigen-binding protein itself. As above, thehalf-life extension domain extends the elimination half-time. Thehalf-life extension domain also alters pharmacodynamic propertiesincluding alteration of tissue distribution, penetration, and diffusionof the trispecific antigen-binding protein. In some embodiments, thehalf-life extension domain provides for improved tissue (includingtumor) targeting, tissue distribution, tissue penetration, diffusionwithin the tissue, and enhanced efficacy as compared with a proteinwithout an half-life extension domain. In one embodiment, therapeuticmethods effectively and efficiently utilize a reduced amount of thetrispecific antigen-binding protein, resulting in reduced side effects,such as reduced non-tumor cell cytotoxicity.

Further, the binding affinity of the half-life extension domain can beselected so as to target a specific elimination half-time in aparticular trispecific antigen-binding protein. Thus, in someembodiments, the half-life extension domain has a high binding affinity.In other embodiments, the half-life extension domain has a mediumbinding affinity. In yet other embodiments, the half-life extensiondomain has a low or marginal binding affinity. Exemplary bindingaffinities include K_(D) concentrations at 10 nM or less (high), between10 nM and 100 nM (medium), and greater than 100 nM (low). As above,binding affinities to HSA are determined by known methods such asSurface Plasmon Resonance (SPR).

In some embodiments, HSA binding domains described herein comprise apolypeptide having a sequence described in Table 8 (SEQ ID NO: 89-112)and subsequences thereof. In some embodiments, the HSA binding domaincomprises a polypeptide having at least 70%-95% or more homology to asequence described in Table 8 (SEQ ID NO: 89-112). In some embodiments,the HSA binding domain comprises a polypeptide having at least 70%, 75%,80%, 85%, 90%, 95%, or more homology to a sequence described in Table 8(SEQ ID NO: 89-112). In some embodiments, the HSA binding domain has asequence comprising at least a portion of a sequence described in Table8 (SEQ ID NO: 89-112). In some embodiments, the HSA binding domaincomprises a polypeptide comprising one or more of the sequencesdescribed in Table 8 (SEQ ID NO: 89-112).

In some embodiments, HSA binding domains described herein comprise asingle domain antibody with a CDR1 comprising SE ID NO: 96, and 99-101.In some embodiments, HSA binding domains described herein comprise asingle domain antibody with a CDR1 comprising SE ID NO: 97, and 102-107.In some embodiments, HSA binding domains described herein comprise asingle domain antibody with a CDR1 comprising SE ID NO: 98, 108 and 109.

Prostate Specific Membrane Antigen (PSMA) Binding Domain

Prostate specific membrane antigen (PSMA) is a 100 kD Type II membraneglycoprotein expressed in prostate tissues having sequence identity withthe transferrin receptor with NAALADase activity. PSMA is expressed inincreased amounts in prostate cancer, and elevated levels of PSMA arealso detectable in the sera of these patients. PSMA expression increaseswith disease progression, becoming highest in metastatic,hormone-refractory disease for which there is no present therapy.

In addition to the described CD3 and half-life extension domains, thePSMA targeting trispecific proteins described herein also comprise adomain that binds to PSMA. The design of the PSMA targeting trispecificproteins described herein allows the binding domain to PSMA to beflexible in that the binding domain to PSMA can be any type of bindingdomain, including but not limited to, domains from a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody. In some embodiments, the binding domainto PSMA is a single chain variable fragments (scFv), single-domainantibody such as a heavy chain variable domain (VH), a light chainvariable domain (VL) and a variable domain (VHH) of camelid derivedsingle domain antibody. In other embodiments, the binding domain to PSMAis a non-Ig binding domain, i.e., antibody mimetic, such as anticalins,affilins, affibody molecules, affimers, affitins, alphabodies, avimers,DARPins, fynomers, kunitz domain peptides, and monobodies. In furtherembodiments, the binding domain to PSMA is a ligand or peptide thatbinds to or associates with PSMA. In yet further embodiments, thebinding domain to PSMA is a knottin. In yet further embodiments, thebinding domain to PSMA is a small molecular entity.

In some embodiments, the PSMA binding domain comprises the followingformula: f1-r1-f2-r2-f3-r3-f4, wherein r1, r2, and r3 arecomplementarity determining regions CDR1, CDR2, and CDR3, respectively,and f1, f2, f3, and f4 are framework residues, and wherein r1 comprisesSEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, or SEQ ID NOL 125, r2comprises SEQ ID No. 117, SEQ ID NO. 118, SEQ ID No. 119, SEQ ID No.120, SEQ ID No. 121, SEQ ID No. 122, SEQ ID No. 123, or SEQ ID NO: 126,and r3 comprises SEQ ID No. 124, or SEQ ID NO: 127.

In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, andCDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQID No. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX4X₅TDYAEX6VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY). In someembodiments, the amino acid residues X₁, X₂, X₃, X₄, X₅, X₆, and X₇ areindependently selected from glutamic acid, proline, serine, histidine,threonine, aspartic acid, glycine, lysine, threonine, glutamine, andtyrosine. In some embodiments, X₁ is proline. In some embodiments, X₂ ishistidine. In some embodiments, X₃ is aspartic acid. In someembodiments, X₄ is lysine. In some embodiments, X₅ is glutamine. In someembodiments, X₆ is tyrosine. In some embodiments, X₇ is serine. The PSMAbinding protein of the present disclosure may in some embodimentscomprise CDR1, CDR2, and CDR3 sequences wherein X₁ is glutamic acid, X₂is histidine, X₃ is aspartic acid, X₄ is glycine, X₅ is threonine, X₆ isserine, and X₇ is serine.

In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, andCDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQID No. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X1is proline. In some embodiments, the PSMA binding domain comprises aCDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is asset forth in SEQ ID No. 162 (RFMISX₁YX₂MH), (b) the amino acid sequenceof CDR2 is as set forth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164(DX₇YGY), wherein X5 is glutamine. In some embodiments, the PSMA bindingdomain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acidsequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX₁YX₂MH), (b)the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163(X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acid sequence of CDR3 is asset forth in SEQ ID No. 164 (DX₇YGY), wherein X₆ is tyrosine. In someembodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3,wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ IDNo. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X₄is lysine, and X₇ is serine. In some embodiments, the PSMA bindingdomain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acidsequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX₁YX₂MH), (b)the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163(X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acid sequence of CDR3 is asset forth in SEQ ID No. 164 (DX₇YGY), wherein X₂ is histidine, X₃ isaspartic acid, X₄ is lysine, and X₇ is serine. In some embodiments, thePSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) theamino acid sequence of CDR1 is as set forth in SEQ ID No. 162(RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as set forth inSEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acid sequenceof CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X₁ isproline, X₂ is histidine, X₃ is aspartic acid, and X₇ is serine. In someembodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3,wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ IDNo. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X₂is histidine, X₃ is aspartic acid, X₅ is glutamine, and X₇ is serine. Insome embodiments, the PSMA binding domain comprises a CDR1, CDR2, andCDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQID No. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X₂is histidine, X₃ is aspartic acid, X₆ is tyrosine, and X₇ is serine. Insome embodiments, the PSMA binding domain comprises a CDR1, CDR2, andCDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQID No. 162 (RFMISX₁YX₂MH), (b) the amino acid sequence of CDR2 is as setforth in SEQ ID No. 163 (X₃INPAX₄X₅TDYAEX₆VKG), and (c) the amino acidsequence of CDR3 is as set forth in SEQ ID No. 164 (DX₇YGY), wherein X₂is histidine, X₃ is aspartic acid, and X₇ is serine.

The PSMA binding domain of the present disclosure may in someembodiments comprise CDR1, CDR2, and CDR3 sequences wherein X₁ isglutamic acid, X₂ is histidine, X₃ is threonine, X₄ is glycine, X₅ isthreonine, X₆ is serine, and X₇ is serine. The PSMA binding domain ofthe present disclosure may in some embodiments comprise CDR1, CDR2, andCDR3 sequences wherein X₁ is glutamic acid, X₂ is histidine, X₃ isthreonine, X₄ is glycine, X₅ is threonine, X₆ is serine, and X₇ isserine. The PSMA binding domain of the present disclosure may in someembodiments comprise CDR1, CDR2, and CDR3 sequences wherein X₁ isglutamic acid, X₂ is serine, X₃ is threonine, X₄ is lysine, X₅ isthreonine, X₆ is serine, and X₇ is serine. The PSMA binding domain ofthe present disclosure may in some embodiments comprise CDR1, CDR2, andCDR3 sequences wherein X₁ is proline, X₂ is serine, X₃ is threonine, X₄is glycine, X₅ is threonine, X6 is serine, and X7 is glycine. The PSMAbinding domain of the present disclosure may in some embodimentscomprise CDR1, CDR2, and CDR3 sequences wherein X₁ is glutamic acid, X₂is serine, X₃ is threonine, X₄ is glycine, X₅ is glutamine, X₆ isserine, and X₇ is glycine. The PSMA binding domain of the presentdisclosure may in some embodiments comprise CDR1, CDR2, and CDR3sequences wherein X₁ is glutamic acid, X₂ is serine, X₃ is threonine, X₄is glycine, X₅ is threonine, X₆ is tyrosine, and X₇ is glycine. The PSMAbinding domain of the present disclosure may in some embodimentscomprise CDR1, CDR2, and CDR3 sequences wherein X₁ is glutamic acid, X₂is histidine, X₃ is aspartic acid, X₄ is lysine, X₅ is threonine, X₆ isserine, and X₇ is serine. The PSMA binding domain of the presentdisclosure may in some embodiments comprise CDR1, CDR2, and CDR3sequences wherein X₁ is proline, X₂ is histidine, X₃ is aspartic acid,X₄ is glycine, X₅ is threonine, X₆ is serine, and X₇ is serine. The PSMAbinding domain of the present disclosure may in some embodimentscomprise CDR1, CDR2, and CDR3 sequences wherein X₁ is glutamic acid, X₂is histidine, X₃ is aspartic acid, X₄ is glutamine, X₅ is threonine, X₆is serine, and X₇ is serine. The PSMA binding domain of the presentdisclosure may in some embodiments comprise CDR1, CDR2, and CDR3sequences wherein X₁ is glutamic acid, X₂ is histidine, X₃ is asparticacid, X₄ is glycine, X₅ is threonine, X₆ is tyrosine, and X₇ is serine.The PSMA binding domain of the present disclosure may in someembodiments comprise CDR1, CDR2, and CDR3 sequences wherein X₂ ishistidine, and X₇ is serine. Exemplary framework sequences are disclosedas SEQ ID NO: 165-168.

In some embodiments, PSMA binding domains described herein comprise apolypeptide having a sequence described in Table 9 (SEQ ID NO: 113-140)and subsequences thereof. In some embodiments, the HSA binding domaincomprises a polypeptide having at least 70%-95% or more homology to asequence described in Table 9 (SEQ ID NO: 113-140). In some embodiments,the HSA binding domain comprises a polypeptide having at least 70%, 75%,80%, 85%, 90%, 95%, or more homology to a sequence described in Table 9(SEQ ID NO: 113-140). In some embodiments, the HSA binding domain has asequence comprising at least a portion of a sequence described in Table9 (SEQ ID NO: 113-140). In some embodiments, the HSA binding domaincomprises a polypeptide comprising one or more of the sequencesdescribed in Table 9 (SEQ ID NO: 113-140).

In some embodiments, PSMA binding domains described herein comprise asingle domain antibody with a CDR1 comprising SE ID NO: 114-116, and125. In some embodiments, PSMA binding domains described herein comprisea single domain antibody with a CDR1 comprising SEQ ID NO: 117-123, and126. In some embodiments, PSMA binding domains described herein comprisea single domain antibody with a CDR1 comprising SE ID NO: 124 and 127.

PSMA Trispecific Protein Modifications

The PSMA targeting trispecific proteins described herein encompassderivatives or analogs in which (i) an amino acid is substituted with anamino acid residue that is not one encoded by the genetic code, (ii) themature polypeptide is fused with another compound such as polyethyleneglycol, or (iii) additional amino acids are fused to the protein, suchas a leader or secretory sequence or a sequence for purification of theprotein.

Typical modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Modifications are made anywhere in PSMA targeting trispecific proteinsdescribed herein, including the peptide backbone, the amino acidside-chains, and the amino or carboxyl termini. Certain common peptidemodifications that are useful for modification of PSMA targetingtrispecific proteins include glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation, blockageof the amino or carboxyl group in a polypeptide, or both, by a covalentmodification, and ADP-ribosylation.

Polynucleotides Encoding PSMA Targeting Trispecific Proteins

Also provided, in some embodiments, are polynucleotide moleculesencoding a PSMA trispecific antigen-binding protein described herein. Insome embodiments, the polynucleotide molecules are provided as a DNAconstruct. In other embodiments, the polynucleotide molecules areprovided as a messenger RNA transcript.

The polynucleotide molecules are constructed by known methods such as bycombining the genes encoding the three binding domains either separatedby peptide linkers or, in other embodiments, directly linked by apeptide bond, into a single genetic construct operably linked to asuitable promoter, and optionally a suitable transcription terminator,and expressing it in bacteria or other appropriate expression systemsuch as, for example CHO cells. In the embodiments where the PSMAbinding domain is a small molecule, the polynucleotides contain genesencoding the CD3 binding domain and the half-life extension domain. Inthe embodiments where the half-life extension domain is a smallmolecule, the polynucleotides contain genes encoding the domains thatbind to CD3 and PSMA. Depending on the vector system and host utilized,any number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. The promoter isselected such that it drives the expression of the polynucleotide in therespective host cell.

In some embodiments, the polynucleotide is inserted into a vector,preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed according to known methods.Vectors of particular interest include plasmids, phagemids, phagederivatives, virii (e.g., retroviruses, adenoviruses, adeno-associatedviruses, herpes viruses, lentiviruses, and the like), and cosmids.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide encoding the polypeptide of the describedtrispecific antigen-binding protein. Examples of expression vectors forexpression in E. coli are pSKK (Le Gall et al., J Immunol Methods.(2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammaliancells.

Thus, the PSMA targeting trispecific proteins as described herein, insome embodiments, are produced by introducing a vector encoding theprotein as described above into a host cell and culturing said host cellunder conditions whereby the protein domains are expressed, may beisolated and, optionally, further purified.

Pharmaceutical Compositions

Also provided, in some embodiments, are pharmaceutical compositionscomprising a PSMA trispecific antigen-binding protein described herein,a vector comprising the polynucleotide encoding the polypeptide of thePSMA targeting trispecific proteins or a host cell transformed by thisvector and at least one pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” includes, but is not limited to,any carrier that does not interfere with the effectiveness of thebiological activity of the ingredients and that is not toxic to thepatient to whom it is administered. Examples of suitable pharmaceuticalcarriers are well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Such carriers can beformulated by conventional methods and can be administered to thesubject at a suitable dose. Preferably, the compositions are sterile.These compositions may also contain adjuvants such as preservative,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents.

In some embodiments of the pharmaceutical compositions, the PSMAtargeting trispecific proteins described herein are encapsulated innanoparticles. In some embodiments, the nanoparticles are fullerenes,liquid crystals, liposome, quantum dots, superparamagneticnanoparticles, dendrimers, or nanorods. In other embodiments of thepharmaceutical compositions, the PSMA trispecific antigen-bindingprotein is attached to liposomes. In some instances, the PSMAtrispecific antigen-binding protein are conjugated to the surface ofliposomes. In some instances, the PSMA trispecific antigen-bindingprotein are encapsulated within the shell of a liposome. In someinstances, the liposome is a cationic liposome.

The PSMA targeting trispecific proteins described herein arecontemplated for use as a medicament. Administration is effected bydifferent ways, e.g. by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. In someembodiments, the route of administration depends on the kind of therapyand the kind of compound contained in the pharmaceutical composition.The dosage regimen will be determined by the attending physician andother clinical factors. Dosages for any one patient depends on manyfactors, including the patient's size, body surface area, age, sex, theparticular compound to be administered, time and route ofadministration, the kind of therapy, general health and other drugsbeing administered concurrently. An “effective dose” refers to amountsof the active ingredient that are sufficient to affect the course andthe severity of the disease, leading to the reduction or remission ofsuch pathology and may be determined using known methods.

Methods of Treatment

Also provided herein, in some embodiments, are methods and uses forstimulating the immune system of an individual in need thereofcomprising administration of a PSMA targeting trispecific proteindescribed herein. In some instances, the administration of a PSMAtargeting trispecific protein described herein induces and/or sustainscytotoxicity towards a cell expressing PSMA. In some instances, the cellexpressing PSMA is a cancer cell.

Also provided herein are methods and uses for a treatment of a disease,disorder or condition associated with PSMA comprising administering toan individual in need thereof a PSMA targeting trispecific proteindescribed herein. Diseases, disorders or conditions associated with PSMAinclude, but are not limited to, a proliferative disease or a tumorousdisease. In one embodiment, the disease, disorder or conditionassociated with PSMA is prostate cancer. In another embodiment, thedisease, disorder, or condition associated with PSMA is renal cancer.

In some embodiments, the prostate cancer is an advanced stage prostatecancer. In some embodiments, the prostate cancer is drug resistant. Insome embodiments, the prostate cancer is anti-androgen drug resistant.In some embodiments, the prostate cancer is metastatic. In someembodiments, the prostate cancer is metastatic and drug resistant (e.g.,anti-androgen drug resistant). In some embodiments, the prostate canceris castration resistant. In some embodiments, the prostate cancer ismetastatic and castration resistant. In some embodiments, the prostatecancer is enzalutamide resistant. In some embodiments, the prostatecancer is enzalutamide and arbiraterone resistant. In some embodiments,the prostate cancer is enzalutamide, arbiraterone, and bicalutamideresistant. In some embodiments, the prostate cancer is docetaxelresistant. In some of these embodiments, the prostate cancer isenzalutamide, arbiraterone, bicalutamide, and docetaxel resistant.

In some embodiments, administering a PSMA targeting trispecific proteindescribed herein inhibits prostate cancer cell growth; inhibits prostatecancer cell migration; inhibits prostate cancer cell invasion;ameliorates the symptoms of prostate cancer; reduces the size of aprostate cancer tumor; reduces the number of prostate cancer tumors;reduces the number of prostate cancer cells; induces prostate cancercell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other celldeath; or enhances the therapeutic effects of a compound selected fromthe group consisting of enzalutamide, abiraterone, docetaxel,bicalutamide, and any combinations thereof.

In some embodiments, the method comprises inhibiting prostate cancercell growth by administering a PSMA targeting trispecific proteindescribed herein. In some embodiments, the method comprises inhibitingprostate cancer cell migration by administering a PSMA targetingtrispecific protein described herein. In some embodiments, the methodcomprises inhibiting prostate cancer cell invasion by administering aPSMA targeting trispecific protein described herein. In someembodiments, the method comprises ameliorating the symptoms of prostatecancer by administering a PSMA targeting trispecific protein describedherein. In some embodiments, the method comprises reducing the size of aprostate cancer tumor by administering a PSMA targeting trispecificprotein described herein. In some embodiments, the method comprisesreducing the number of prostate cancer tumors by administering a PSMAtargeting trispecific protein described herein. In some embodiments, themethod comprises reducing the number of prostate cancer cells byadministering a PSMA targeting trispecific protein described herein. Insome embodiments, the method comprises inducing prostate cancer cellnecrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell deathby administering a PSMA targeting trispecific protein described herein.

As used herein, in some embodiments, “treatment” or “treating” or“treated” refers to therapeutic treatment wherein the object is to slow(lessen) an undesired physiological condition, disorder or disease, orto obtain beneficial or desired clinical results. For the purposesdescribed herein, beneficial or desired clinical results include, butare not limited to, alleviation of symptoms; diminishment of the extentof the condition, disorder or disease; stabilization (i.e., notworsening) of the state of the condition, disorder or disease; delay inonset or slowing of the progression of the condition, disorder ordisease; amelioration of the condition, disorder or disease state; andremission (whether partial or total), whether detectable orundetectable, or enhancement or improvement of the condition, disorderor disease. Treatment includes eliciting a clinically significantresponse without excessive levels of side effects. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment. In other embodiments, “treatment” or “treating” or“treated” refers to prophylactic measures, wherein the object is todelay onset of or reduce severity of an undesired physiologicalcondition, disorder or disease, such as, for example is a person who ispredisposed to a disease (e.g., an individual who carries a geneticmarker for a disease such as prostate cancer).

In some embodiments of the methods described herein, the PSMA targetingtrispecific proteins are administered in combination with an agent fortreatment of the particular disease, disorder or condition. Agentsinclude but are not limited to, therapies involving antibodies, smallmolecules (e.g., chemotherapeutics), hormones (steroidal, peptide, andthe like), radiotherapies (γ-rays, X-rays, and/or the directed deliveryof radioisotopes, microwaves, UV radiation and the like), gene therapies(e.g., antisense, retroviral therapy and the like) and otherimmunotherapies. In some embodiments, the PSMA targeting trispecificproteins are administered in combination with anti-diarrheal agents,anti-emetic agents, analgesics, opioids and/or non-steroidalanti-inflammatory agents. In some embodiments, the PSMA targetingtrispecific proteins are administered before, during, or after surgery.

Certain Definitions

As used herein, “elimination half-time” is used in its ordinary sense,as is described in Goodman and Gillman's The Pharmaceutical Basis ofTherapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and AlfredGilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass aquantitative measure of the time course of drug elimination. Theelimination of most drugs is exponential (i.e., follows first-orderkinetics), since drug concentrations usually do not approach thoserequired for saturation of the elimination process. The rate of anexponential process may be expressed by its rate constant, k, whichexpresses the fractional change per unit of time, or by its half-time,t_(1/2) the time required for 50% completion of the process. The unitsof these two constants are time⁻ time, respectively. A first-order rateconstant and the half-time of the reaction are simply related(k×t_(1/2)=0.693) and may be interchanged accordingly. Since first-orderelimination kinetics dictates that a constant fraction of drug is lostper unit time, a plot of the log of drug concentration versus time islinear at all times following the initial distribution phase (i.e. afterdrug absorption and distribution are complete). The half-time for drugelimination can be accurately determined from such a graph.

As used herein, the phrase “prostate cancer” or “advanced stage prostatecancer” includes a class of prostate cancers that has progressed beyondearly stages of the disease. Typically, advanced stage prostate cancersare associated with a poor prognosis. Types of advanced stage prostatecancers include, but are not limited to, metastatic prostate cancer,drug-resistant prostate cancer such as anti-androgen-resistant prostatecancer (e.g., enzalutamide-resistant prostate cancer,abiraterone-resistant prostate cancer, bicalutamide-resistant prostatecancer, and the like), hormone refractory prostate cancer,castration-resistant prostate cancer, metastatic castration-resistantprostate cancer, docetaxel-resistant prostate cancer, androgen receptorsplice variant-7 (AR-V7)-induced drug-resistant prostate cancer such asAR-V7-induced anti-androgen-resistant prostate cancer (e.g.,AR-V7-induced enzalutamide-resistant prostate cancer), aldo-ketoreductase family 1 member C3 (AKR1C3)-induced drug-resistant prostatecancer such as AKR1C3-induced anti-androgen-resistant prostate cancer(e.g., AKR1C3-induced enzalutamide-resistant prostate cancer), andcombinations thereof. In some instances, the advanced stage prostatecancers do not generally respond, or are resistant, to treatment withone or more of the following conventional prostate cancer therapies:enzalutamide, arbiraterone, bicalutamide, and docetaxel. Compounds,compositions, and methods of the present disclosure are provided fortreating prostate cancer, such as advanced stage prostate cancer,including any one or more (e.g., two, three, four, five, six, seven,eight, nine, ten, or more) of the types of advanced stage prostatecancers disclosed herein.

EXAMPLES Example 1: Methods to Assess Binding and Cytotoxic Activitiesof Trispecific Antigen Binding Molecules

Protein Production

Sequences of trispecific molecules were cloned into mammalian expressionvector pcDNA 3.4 (Invitrogen) preceded by a leader sequence and followedby a 6× Histidine Tag (SEQ ID NO: 161). Expi293F cells (LifeTechnologies A14527) were maintained in suspension in Optimum GrowthFlasks (Thomson) between 0.2 to 8×1e6 cells/ml in Expi293 media.Purified plasmid DNA was transfected into Expi293 cells in accordancewith Expi293 Expression System Kit (Life Technologies, A14635)protocols, and maintained for 4-6 days post transfection. Conditionedmedia was partially purified by affinity and desalting chromatography.Trispecific proteins were subsequently polished by ion exchange or,alternatively, concentrated with Amicon Ultra centrifugal filtrationunits (EMD Millipore), applied to Superdex 200 size exclusion media (GEHealthcare) and resolved in a neutral buffer containing excipients.Fraction pooling and final purity were assessed by SDS-PAGE andanalytical SEC.

Affinity Measurements

The affinities of the all binding domains molecules were measured bybiolayer inferometry using an Octet instrument.

PSMA affinities were measured by loading human PSMA-Fc protein (100 nM)onto anti-human IgG Fc biosensors for 120 seconds, followed by a 60second baseline, after which associations were measured by incubatingthe sensor tip in a dilution series of the trispecific molecules for 180seconds, followed by dissociation for 50 seconds. EGFR and CD3affinities were measured by loading human EGFR-Fc protein or humanCD3-Flag-Fc protein, respectively, (100 nM) onto anti-human IgG Fcbiosensors for 120 seconds, followed by a 60 second baseline, afterwhich associations were measured by incubating the sensor tip in adilution series of the trispecific molecules for 180 seconds, followedby dissociation for 300 seconds. Affinities to human serum albumin (HSA)were measured by loading biotinylated albumin onto streptavidinbiosensors, then following the same kinetic parameters as for CD3affinity measurements. All steps were performed at 30° C. in 0.25%casein in phosphate-buffered saline.

Cytotoxicity Assays

A human T-cell dependent cellular cytotoxicity (TDCC) assay was used tomeasure the ability of T cell engagers, including trispecific molecules,to direct T cells to kill tumor cells (Nazarian et al. 2015. J BiomolScreen. 20:519-27). In this assay, T cells and target cancer cell linecells are mixed together at a 10:1 ratio in a 384 wells plate, andvarying amounts of T cell engager are added. After 48 hours, the T cellsare washed away leaving attached to the plate target cells that were notkilled by the T cells. To quantitate the remaining viable cells,CellTiter-Glo® Luminescent Cell Viability Assay (Promega) is used. Insome cases, the target cells are engineered to express luciferase. Inthese cases, viability of the target cells is assessed by performing aluminescent luciferase assay with STEADYGLO® reagent (Promega), whereviability is directly proportional to the amount of luciferase activity.

Stability Assays

The stability of the trispecific binding proteins was assessed at lowconcentrations in the presence of non-human primate serum. TriTACs werediluted to 33 μg/ml in Cynomolgus serum (BioReclamationIVT) and eitherincubated for 2 d at 37° C. or subjected to five freeze/thaw cycles.Following the treatment, the samples were assessed in cytotoxicity(TDCC) assays and their remaining activity was compared to untreatedstock solutions.

Xenograft Assays

The in vivo efficacy of trispecific binding proteins was assessed inxenograft experiments (Crown Bioscience, Taicang). NOD/SCID micedeficient in the common gamma chain (NCG, Model Animal Research Centerof Nanjing University) were inoculated on day 0 with a mixture of 5e622Rv1 human prostate cancer cells and 5e6 resting, human T cells thatwere isolated from a healthy, human donor. The mice were randomized intothree groups, and treated with vehicle, 0.5 mg/kg PSMA TriTAC C324 or0.5 mg/kg PSMA BiTE. Treatments were administered daily for 10 days viai.v. bolus injection. Animals were checked daily for morbidity andmortality. Tumor volumes were determined twice weekly with a caliper.The study was terminated after 30 days.

PK Assays

The purpose of this study was to evaluate the single dosepharmacokinetics of trispecific binding proteins following intravenousinjection. 2 experimentally naïve cynomolgus monkeys per group (1 maleand 1 female) were given compound via a slow IV bolus injectionadministered over approximately 1 minute. Following dose administration,cage side observations were performed once daily and body weights wererecorded weekly. Blood samples were collected and processed to serum forpharmacokinetic analysis through 21 days post dose administration.

Concentrations of test articles were determined from monkey serum withan electroluminescent readout (Meso Scale Diagnostics, Rockville). 96well plates with immobilized, recombinant CD3 were used to capture theanalyte. Detection was performed with sulfo-tagged, recombinant PSMA ona MSD reader according to the manufacturer's instructions.

Example 2: Assessing the Impact of CD3 Affinity on the Properties ofTrispecific Molecules

PSMA targeting trispecific molecules with distinct CD3 binding domainswere studied to demonstrate the effects of altering CD3 affinity. Anexemplary PSMA targeting trispecific molecule is illustrated in FIG. 1.Table 1 lists the affinity of each molecule for the three bindingpartners (PSMA, CD3, HSA). Affinities were measured by biolayerinterferometry using an Octet instrument (Pall Forte Bio). Reduced CD3affinity leads to a loss in potency in terms of T cell mediated cellulartoxicity (FIGS. 2A-C). The pharmacokinetic properties of thesetrispecific molecules was assessed in cynomolgus monkeys. Molecules withhigh affinity for CD3 like TriTAC C236 have a terminal half-life ofapprox. 90 h (FIG. 3). Despite the altered ability to bind CD3 on Tcells, the terminal half-life of two molecules with different CD3affinities shown in FIG. 4 is very similar. However, the reduced CD3affinity appears to lead to a larger volume of distribution, which isconsistent with reduced sequestration of trispecific molecule by Tcells. There were no adverse clinical observations or body weightchanges noted during the study period.

TABLE 1 Binding Affinities for Human and Cynomolgus Antigens anti-PSMAKD value (nM) anti-Albumin KD value (nM) anti-CD3e KD value (nM) ratioratio ratio cyno/ cyno/ cyno/ human cyno hum pHSA CSA hum human cyno humTool TriTAC high 16.3 0 0 22.7 25.4 1.1 6.0 4.7 0.8 aff. - C236 TriTACCD3 high 17.9 0 0 9.8 9.7 1 7.4 5.8 0.8 aff. - C324 TriTAC CD3 med 13.60 0 8.8 8.3 0.9 40.6 33.6 0.8 aff. - C339 TriTAC CD3 low 15.3 0 0 10.19.7 1 217 160 0.7 aff - C325

Example 3: Assessing the Impact of PSMA Affinity on the Properties ofTrispecific Molecules

PSMA targeting trispecific molecules with distinct PSMA binding domainswere studied to demonstrate the effects of altering PSMA affinity. Table2 lists the affinity of each molecule for the three binding partners(PSMA, CD3, HSA). Reduced PSMA affinity leads to a loss in potency interms of T cell mediated cellular toxicity (FIGS. 5A-C).

TABLE 2 Binding Affinities for Human and Cynomolgus Antigens anti-PSMAKD value (nM) anti-Albumin KD value (nM) anti-CD3e KD value (nM) ratioratio ratio cyno/ cyno/ cyno/ human cyno hum pHSA CSA hum human cyno humPSMA-TriTAC 22.0 0 n/a 6.6 6.6 1.0 8.3 4.3 0.52 (p8)-C362 PSMA TriTAC3.7 540 146 7.6 8.4 1.1 8.0 5.2 0.65 (HDS) - C363 PSMA TriTAC 0.15 6634423 8.4 8.6 1.0 7.7 3.8 0.49 (HTS)- C364

Example 4: In Vivo Efficacy of PSMA Targeting Trispecific Molecules

The PSMA targeting trispecific molecule C324 was assessed for itsability to inhibit the growth of tumors in mice. For this experiment,immunocompromised mice reconstituted with human T cells weresubcutaneously inoculated with PSMA expressing human prostate tumorcells (22Rv1) and treated daily for 10 days with 0.5 mg/kg i.v. ofeither PSMA targeting BiTE or TriTAC molecules. Tumor growth wasmeasured for 30. Over the course of the experiment, the trispecificmolecule was able to inhibit tumor growth with an efficacy comparable toa BiTE molecule (FIG. 6).

Example 5: Specificity of Trispecific Molecules

In order to assess the specificity of PSMA targeting TriTAC molecules,their ability to induce T cells to kill tumor cells was tested withtumor cells that are negative for PSMA (FIG. 7A). An EGFR targetingTriTAC molecule served as positive control, a GFP targeting TriTACmolecule as negative control. All three TriTACs with distinct PSMAbinding domains showed the expected activity against the PSMA positivecell line LNCaP (FIG. 7B), but did not reach EC50s in the PSMA negativetumor cell lines KMS12BM and OVCAR8 (FIGS. 7C and 7D). The EC50s aresummarized in Table 3. At very high TriTAC concentrations (>1 nM), somelimited off-target cell killing could be observed for TriTACs C362 andC363, while C364 did not show significant cell killing under any of thetested conditions.

TABLE 3 Cell killing activity of TriTAC molecules in with antigenpositive and negative tumor cell lines (EC50 [pM]) TriTAC LNCaP KMS12BMOVCAR8 PSMA p8 TriTAC C362 13.0 >10,000 >10,000 PSMA HDS TriTAC C3636.2 >10,000 >10,000 PSMA HTS TriTAC C364 0.8 >10,000 >10,000 EGFR TriTACC131 9.4 >10,000 6 GFP TriTAC C >10,000 >10,000 >10,000

Example 6: Stress Tests and Protein Stability

Four PSMA targeting trispecific molecules were either incubated for 48 hin Cynomolgus serum at low concentrations (33.3 μg/ml) or subjected tofive freeze thaw cycles in Cynomolgus serum. After the treatment, thebio-activity of the TriTAC molecules was assessed in cell killing assaysand compared to unstressed samples (“positive control”, FIG. 8A-D). Allmolecules maintained the majority of their cell killing activity. TriTACC362 was the most stress resistant and did not appear to lose anyactivity under the conditions tested here.

Example 7: Xenograft Tumor Model

The PSMA targeting trispecific proteins of the previous examples areevaluated in a xenograft model.

Male immune-deficient NCG mice are subcutaneously inoculated with 5×10⁶22 Rv1 cells into their the right dorsal flank. When tumors reach 100 to200 mm³, animals are allocated into 3 treatment groups. Groups 2 and 3(8 animals each) are intraperitoneally injected with 1.5×10⁷ activatedhuman T-cells. Three days later, animals from Group 3 are subsequentlytreated with a total of 9 intravenous doses of 50 μg PSMA trispecificantigen-binding protein of Example 1 (qdx9d). Groups 1 and 2 are onlytreated with vehicle. Body weight and tumor volume are determined for 30days. It is expected that tumor growth in mice treated with the PSMAtrispecific antigen-binding protein is significantly reduced incomparison to the tumor growth in respective vehicle-treated controlgroup.

Example 8: Proof-of-Concept Clinical Trial Protocol for Administrationof the PSMA Trispecific Antigen-Binding Protein of Example 1 to ProstateCancer Patients

This is a Phase I/II clinical trial for studying the PSMA trispecificantigen-binding protein of Example 1 as a treatment for Prostate Cancer.

Study Outcomes:

Primary: Maximum tolerated dose of PSMA targeting trispecific proteinsof the previous examples

Secondary: To determine whether in vitro response of PSMA targetingtrispecific proteins of is the previous examples are associated withclinical response

Phase I

The maximum tolerated dose (MTD) will be determined in the phase Isection of the trial.

1.1 The maximum tolerated dose (MTD) will be determined in the phase Isection of the trial.1.2 Patients who fulfill eligibility criteria will be entered into thetrial to PSMA targeting trispecific proteins of the previous examples.1.3 The goal is to identify the highest dose of PSMA targetingtrispecific proteins of the previous examples that can be administeredsafely without severe or unmanageable side effects in participants. Thedose given will depend on the number of participants who have beenenrolled in the study prior and how well the dose was tolerated. Not allparticipants will receive the same dose.

Phase II

2.1 A subsequent phase II section will be treated at the MTD with a goalof determining if therapy with therapy of PSMA targeting trispecificproteins of the previous examples results in at least a 20% responserate.Primary Outcome for the Phase II—To determine if therapy of PSMAtargeting trispecific proteins of the previous examples results in atleast 20% of patients achieving a clinical response (blast response,minor response, partial response, or complete response)

Eligibility:

Histologically confirmed newly diagnosed aggressive prostate canceraccording to the current World Health Organisation Classification, from2001 to 2007

Any stage of disease.

Treatment with docetaxel and prednisone (+/−surgery).

Age≥18 years

Karnofsky performance status≥50% or ECOG performance status 0-2

Life expectancy≥6 weeks

Example 9: Activity of an Exemplary PSMA Antigen-Binding Protein (PSMATargeting TriTAC Molecule) in Redirected T Cell Killing Assays Using aPanel of PSMA Expressing Cell Lines and T Cells from Different Donors

This study was carried out to demonstrate that the activity of theexemplary PSMA trispecific antigen-binding protein is not limited toLNCaP cells or a single cell donor.

Redirected T cell killing assays were performed using T cells from fourdifferent donors and the human PSMA-expressing prostate cancer celllines VCaP, LNCaP, MDAPCa2b, and 22Rv1. With one exception, the PSMAtrispecific antigen-binding protein was able to direct killing of thesecancer cell lines using T cells from all donors with EC₅₀ values of 0.2to 1.5 pM, as shown in Table 4. With the prostate cancer cell line 22Rv1 and Donor 24, little to no killing was observed (data not shown).Donor 24 also only resulted approximately 50% killing of the MDAPCa2bcell line whereas T cells from the other 3 donors resulted in almostcomplete killing of this cell line (data not shown). Control assaysdemonstrated that killing by the PSMA trispecific antigen-bindingprotein was PSMA specific. No killing was observed when PSMA-expressingcells were treated with a control trispecific protein targeting greenfluorescent protein (GFP) instead of PSMA (data not shown). Similarly,the PSMA trispecific antigen-binding protein was inactive with celllines that lack PSMA expression, NCI-1563 and HCT116, also shown inTable 4.

TABLE 4 EC₅₀ Values from TDCC Assays with Six Human Cancer Cell Linesand Four Different T Cell Donors TDCC EC₅₀ Values (M) Cell Line Donor 24Donor 8144 Donor 72 Donor 41 LNCaP 1.5E−12 2.2E−13 3.6E−13 4.3E−13MDAPCa2b 4.8E−12 4.1E−13 4.9E−13 6.5E−13 VCaP 6.4E−13 1.6E−13 2.0E−133.5E−13 22Rv1 n/a 7.2E−13 1.4E−12 1.3E−12HCT116 >1.0E−8  >1.0E−8  >1.0E−8  >1.0E−8 NCI-1563 >1.0E−8  >1.0E−8  >1.0E−8  >1.0E−8 

Example 10: Stimulation of Cytokine Expression in by an Exemplary PSMATrispecific Antigen-Binding Protein (PSMA Targeting TriTAC Molecule) inRedirected T Cell Killing Assays

This study was carried out to demonstrate activation of T cells by theexemplary PSMA trispecific antigen-binding protein during redirected Tcell killing assays by measuring secretion of cytokine into the assaymedium by activated T cells.

Conditioned media collected from redirected T cell killing assays, asdescribed above in Example 9, were analyzed for expression of thecytokines TNFα and IFNγ. Cytokines were measured using AlphaLISA assays(Perkin-Elmer). Adding a titration of the PSMA antigen-binding proteinto T cells from four different donors and four PSMA-expressing celllines, LNCaP, VCaP, MDAPCa2b, and 22Rv1 resulted in increased levels ofTNFα. The results for TNFα expression and IFN γ expression levels in theconditioned media are shown in Tables 5 and 6, respectively. The EC₅₀values for the PSMA antigen-binding protein induced expression of thesecytokines ranged from 3 to 15 pM. Increased cytokine levels were notobserved with a control trispecific protein targeting GFP. Similarly,when assays were performed with two cell lines that lack PSMAexpression, HCT116 and NCI-H1563, PSMA HTS TriTAC also did not increaseTNFα or IFNγ expression.

TABLE 5 EC₅₀ Values for TNFα Expression in Media from PSMA TrispecificAntigen-Binding Protein TDCC Assays with Six Human Cancer Cell Lines andT Cells from Four Different Donors Cell Line Donor 24 Donor 8144 Donor41 Donor72 LNCaP 4.9E−12 2.8E−12 4.0E−12 3.2E−12 VCaP 3.2E−12 2.9E−122.9E−12 2.9E−12 MDAPCa2b 2.1E−11 4.0E−12 5.5E−12 3.6E−12 22Rv1 8.9E−122.5E−12 4.0E−12 3.3E−12 HCT116 >1E−8  >1E−8  >1E−8  >1E−8 NCI-H1563 >1E−8  >1E−8  >1E−8  >1E−8 

TABLE 6 EC₅₀ Values for IFNγ Expression in Media from PSMA TrispecificAntigen-Binding Protein TDCC Assays with Six Human Cancer Cell Lines andT Cells from Four Different Donors Cell Line Donor 24 Donor 8144 Donor41 Donor72 LNCaP 4.2E−12 4.2E−12 4.2E−12 2.8E−12 VCaP 5.1E−12 1.5E−113.4E−12 4.9E−12 MDAPCa2b 1.5E−11 5.8E−12 9.7E−12 3.5E−12 22Rv1 7.8E−123.0E−12 9.1E−12 3.0E−12 HCT116 >1E−8  >1E−8  >1E−8  >1E−8 NCI-H1563 >1E−8  >1E−8  >1E−8  >1E−8 

Example 11: Activity of an Exemplary PSMA Trispecific Antigen-BindingProtein (PSMA Targeting TriTAC) in Redirected T Cell Killing Assay(TDCC) Using T Cells from Cynomolgus Monkeys

This study was carried out to test the ability of the exemplary PSMAtrispecific antigen-binding protein to direct T cells from cynomolgusmonkeys to kill PSMA-expressing cell lines.

TDCC assays were set up using peripheral blood mononuclear cells (PBMCs)from cynomolgus monkeys. Cyno PBMCs were added to LNCaP cells at a 10:1ratio. It was observed that the PSMA trispecific antigen-binding proteinredirected killing of LNCaP by the cyno PBMCs with an EC₅₀ value of 11pM. The result is shown in FIG. 9A. To confirm these results, a secondcell line was used, MDAPCa2b, and PBMCs from a second cynomolgus monkeydonor were tested. Redirected killing of the target cells was observedwith an EC₅₀ value of 2.2 pM. The result is shown in FIG. 9B. Killingwas specific to the anti-PMSA arm of the PSMA trispecificantigen-binding protein as killing was not observed with a negativecontrol trispecific protein targeting GFP. These data demonstrate thatthe PSMA antigen-binding trispecific protein can direct cynomolgus Tcells to kill target cells expressing human PSMA.

Example 12: Expression of Markers of T Cell Activation in Redirect TCell Killing Assays with an Exemplary PSMA Trispecific Antigen-BindingProtein (PSMA Targeting TriTAC Molecule)

This study was performed to assess whether T cells were activated whenthe exemplary PSMA trispecific antigen-binding protein directed the Tcells to kill target cells.

The assays were set up using conditions for the redirected T cellkillings assays described in the above example. T cell activation wasassessed by measuring expression of CD25 and CD69 on the surface of theT cells using flow cytometry. The PSMA trispecific antigen-bindingprotein was added to a 10:1 mixture of purified human T cells and theprostate cancer cell line VCaP. Upon addition of increasing amounts ofthe PSMA trispecific antigen-binding protein, increased CD69 expressionand CD25 expression was observed, as shown in FIG. 10. EC₅₀ value was0.3 pM for CD69 and 0.2 pM for CD25. A trispecific protein targeting GFPwas included in these assays as negative control, and little to noincrease in CD69 or CD25 expression is observed with the GFP targetingtrispecific protein, also shown in FIG. 10.

Example 13: Stimulation of T Cell Proliferation by an Exemplary PSMATrispecific Antigen-Binding Protein (PSMA Targeting TriTAC Molecule) inthe Presence of PSMA Expressing Target Cells

This study was used as an additional method to demonstrate that theexemplary PSMA trispecific antigen-binding protein was able to activateT cells when it redirects them to kill target cells.

T cell proliferation assays were set up using the conditions of the Tcell redirected killing assay using LNCaP target cells, as describedabove, and measuring the number of T cells present at 72 hours. Theexemplary PSMA trispecific antigen-binding protein stimulatedproliferation with an EC₅₀ value of 0.5 pM. As negative control, atrispecific protein targeting GFP was included in the assay, and noincreased proliferation was observed with this protein. The results forthe T cell proliferation assay are illustrated in FIG. 11.

Example 14: Redirected T Cell Killing of LNCaP Cells by Three ExemplaryPSMA Trispecific Antigen-Binding Proteins (PSMA Targeting TriTACMolecules PH1T, PH, and Z2)

This study was carried out to test the ability of three exemplary PSMAtrispecific antigen-binding proteins, having the sequences as set forthin SEQ ID Nos: 150, 151, and 152, to redirect T cells to kill the LNCaPcell line.

In TDCC assays, set up as described in above examples, the PSMA PH1TTriTAC (SEQ ID No: 150) and PSMA PH1 TriTAC (SEQ ID NO: 151) proteinsdirected killing with EC₅₀ values of 25 and 20 pM, respectively, asshown in FIG. 12A; and the PSMA Z2 TriTAC (SEQ ID NO: 152) proteindirected killing with an EC₅₀ value of 0.8 pM, as shown in FIG. 12B.

TABLE 7 CD3 Binding Domain Sequences SEQ ID NO: Description AA Sequence1 Anti-CD3, clone 2B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCTLWYSNRWVFGGGTKLTVL 2Anti-CD3, clone 9F2 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNKYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYDNRWVFGGGTKLTVL3 Anti-CD3, clone 5A2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSHISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGYVTSGNYPNWVQQKPGQAPRGLIGGTSFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWIFGGGTKLTVL4 Anti-CD3, clone 6A2 EVQLVESGGGLVQPGGSLKLSCAASGFMFNKYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWATWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQAPRGLIGGTKLLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNSWVFGGGTKLTVL5 Anti-CD3, clone 2D2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYKDSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL6 Anti-CD3, clone 3F2 EVQLVESGGGLVQPGGSLKLSCAASGFTYNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSKGAVTSGNYPNWVQQKPGQAPRGLIGGTKELAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCTLWYSNRWVFGGGTKLTVL7 Anti-CD3, clone 1A2 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHTNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTYFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL8 Anti-CD3, clone 1C2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADAVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSQISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTDGNYPNWVQQKPGQAPRGLIGGIKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL9 Anti-CD3, clone 2E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAVNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGESTGAVTSGNYPNWVQQKPGQAPRGLIGGTKILAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL10 Anti-CD3, clone 10E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYPMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKNEDTAVYYCVRHGNFNNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTKGNYPNWVQQKPGQAPRGLIGGTKMLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCALWYSNRWVFGGGTKLTVL11 Anti-CD3, clone 2H2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL12 Anti-CD3, clone 2A4 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGDSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTHGNYPNWVQQKPGQAPRGLIGGTKVLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVL13 Anti-CD3, clone 10B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKGLEWVARIRSGYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSYTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFNAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYANRWVFGGGTKLTVL14 Anti-CD3, clone 1G4 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSLISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSSGAVTSGNYPNWVQQKPGQAPRGLIGGTKFGAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL15 wt anti-CD3 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL16 wt anti-CD3 HC CDR1 GFTFNKYAMN 17 wt anti-CD3 HC CDR2RIRSKYNNYATYYADSVK 18 wt anti-CD3 HC CDR3 HGNFGNSYISYWAY 19wt anti-CD3 LC CDR1 GSSTGAVTSGNYPN 20 wt anti-CD3 LC CDR2 GTKFLAP 21wt anti-CD3 LC CDR3 VLWYSNRWV 22 HC CDR1 variant 1 GNTFNKYAMN 23HC CDR1 variant 2 GFEFNKYAMN 24 HC CDR1 variant 3 GFMFNKYAMN 25HC CDR1 variant 4 GFTYNKYAMN 26 HC CDR1 variant 5 GFTFNNYAMN 27HC CDR1 variant 6 GFTFNGYAMN 28 HC CDR1 variant 7 GFTFNTYAMN 29HC CDR1 variant 8 GFTFNEYAMN 30 HC CDR1 variant 9 GFTFNKYPMN 31HC CDR1 variant 10 GFTFNKYAVN 32 HC CDR1 variant 11 GFTFNKYAIN 33HC CDR1 variant 12 GFTFNKYALN 34 HC CDR2 variant 1 RIRSGYNNYATYYADSVK 35HC CDR2 variant 2 RIRSKSNNYATYYADSVK 36 HC CDR2 variant 3RIRSKYNKYATYYADSVK 37 HC CDR2 variant 4 RIRSKYNNYETYYADSVK 38HC CDR2 variant 5 RIRSKYNNYATEYADSVK 39 HC CDR2 variant 6RIRSKYNNYATYYKDSVK 40 HC CDR2 variant 7 RIRSKYNNYATYYADEVK 41HC CDR2 variant 8 RIRSKYNNYATYYADAVK 42 HC CDR2 variant 9RIRSKYNNYATYYADQVK 43 HC CDR2 variant 10 RIRSKYNNYATYYADDVK 44HC CDR3 variant 1 HANFGNSYISYWAY 45 HC CDR3 variant 2 HTNFGNSYISYWAY 46HC CDR3 variant 3 HGNFNNSYISYWAY 47 HC CDR3 variant 4 HGNFGDSYISYWAY 48HC CDR3 variant 5 HGNFGNSHISYWAY 49 HC CDR3 variant 6 HGNFGNSPISYWAY 50HC CDR3 variant 7 HGNFGNSQISYWAY 51 HC CDR3 variant 8 HGNFGNSLISYWAY 52HC CDR3 variant 9 HGNFGNSGISYWAY 53 HC CDR3 variant 10 HGNFGNSYISYWAT 54LC CDR1 variant 1 ASSTGAVTSGNYPN 55 LC CDR1 variant 2 GESTGAVTSGNYPN 56LC CDR1 variant 3 GSYTGAVTSGNYPN 57 LC CDR1 variant 4 GSSFGAVTSGNYPN 58LC CDR1 variant 5 GSSKGAVTSGNYPN 59 LC CDR1 variant 6 GSSSGAVTSGNYPN 60LC CDR1 variant 7 GSSTGYVTSGNYPN 61 LC CDR1 variant 8 GSSTGAVVSGNYPN 62LC CDR1 variant 9 GSSTGAVTDGNYPN 63 LC CDR1 variant 10 GSSTGAVTKGNYPN 64LC CDR1 variant 11 GSSTGAVTHGNYPN 65 LC CDR1 variant 12 GSSTGAVTVGNYPN66 LC CDR1 variant 13 GSSTGAVTSGYYPN 67 LC CDR2 variant 1 GIKFLAP 68LC CDR2 variant 2 GTEFLAP 69 LC CDR2 variant 3 GTYFLAP 70LC CDR2 variant 4 GTSFLAP 71 LC CDR2 variant 5 GTNFLAP 72LC CDR2 variant 6 GTKLLAP 73 LC CDR2 variant 7 GTKELAP 74LC CDR2 variant 8 GTKILAP 75 LC CDR2 variant 9 GTKMLAP 76LC CDR2 variant 10 GTKVLAP 77 LC CDR2 variant 11 GTKFNAP 78LC CDR2 variant 12 GTKFGAP 79 LC CDR2 variant 13 GTKFLVP 80LC CDR3 variant 1 TLWYSNRWV 81 LC CDR3 variant 2 ALWYSNRWV 82LC CDR3 variant 3 VLWYDNRWV 83 LC CDR3 variant 4 VLWYANRWV 84LC CDR3 variant 5 VLWYSNSWV 85 LC CDR3 variant 6 VLWYSNRWI 86LC CDR3 variant 7 VLWYSNRWA 87 Anti-CD3, clone 2G5EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYALNWVRQAPGKGLEWVARIRSKYNNYATEYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTNFLAPGTPERFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWAFGGGTKLTVL88 Anti-CD3, clone 8A5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNEYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADDVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSGISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTVGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL

TABLE 8 HSA Binding Domain Sequences SEQ ID NO: Description AA Sequence89 Anti-HSA sdAb clone 6C EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS90 Anti-HSA sdAb clone 7A EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGADTLYADSLKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSKSSQGTLVTVSS91 Anti-HSA sdAb clone 7G EVQLVESGGGLVQPGNSLRLSCAASGFTYSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSKSSQGTLVTVSS92 Anti-HSA sdAb clone 8H EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGTDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSS93 Anti-HSA sdAb clone 9A EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSKSSQGTLVTVSS94 Anti-HSA sdAb clone 10G EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSVSSQGTLVTVSS95 wt anti-HSA EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS96 wt anti-HSA CDR1 GFTFSSFGMS 97 wt anti-HSA CDR2 SISGSGSDTLYADSVK 98wt anti-HSACDR3 GGSLSR 99 CDR1 variant 1 GFTFSRFGMS 100 CDR1 variant 2GFTFSKFGMS 101 CDR1 variant 3 GFTYSSFGMS 102 CDR2 variant 1SISGSGADTLYADSLK 103 CDR2 variant 2 SISGSGTDTLYADSVK 104 CDR2 variant 3SISGSGRDTLYADSVK 105 CDR2 variant 4 SISGSGSDTLYAESVK 106 CDR2 variant 5SISGSGTDTLYAESVK 107 CDR2 variant 6 SISGSGRDTLYAESVK 108 CDR3 variant 1GGSLSK 109 CDR3 variant 2 GGSLSV 110 Anti-HSA sdAb clone 6CEEVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWVRQAPGKGLEWVSSISGSGSDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS111 Anti-HSA sdAb clone 8HEEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGTDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS112 Anti-HSA sdAb clone 10GEEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSVSSQGTLVTVSS

TABLE 9 PSMA Binding Domain Sequences SEQ ID NO: Description AA Sequence113 wt anti-PSMA EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCDGYGYRGQGTQVTVSS 114CDR1 variant 1 RFMISEYHMH 115 CDR1 variant 2 RFMISPYSMH 116CDR1 variant 3 RFMISPYHMH 117 CDR2 variant 1 DINPAGTTDYAESVKG 118CDR2 variant 2 TINPAKTTDYAESVKG 119 CDR2 variant 3 TINPAGQTDYAESVKG 120CDR2 variant 4 TINPAGTTDYAEYVKG 121 CDR2 variant 5 DINPAKTTDYAESVKG 122CDR2 variant 6 DINPAGQTDYAESVKG 123 CDR2 variant 7 DINPAGTTDYAEYVKG 124CDR3 variant 1 DSYGY 125 CDR1 variant 4 RFMISEYSMH 126 CDR2 variant 8TINPAGTTDYAESVKG 127 CDR3 variant 2 DGYGY 128 Anti-PSMA clone 1EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDGYGYRGQGTLVTVSS 129Anti-PSMA clone 2 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWVRQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDSYGYRGQGTLVTVSS 130Anti-PSMA clone 3 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDSYGYRGQGTLVTVSS 131Anti-PSMA clone 4 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAKTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDSYGYRGQGTLVTVSS 132Anti-PSMA clone 5 EVQLVESGGGLVQPGGSLRLSCAASRFMISPYSMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDGYGYRGQGTLVTVSS 133Anti-PSMA clone 6 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGQTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDGYGYRGQGTLVTVSS 134Anti-PSMA clone 7 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGKGLEWVSTINPAGTTDYAEYVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDGYGYRGQGTLVTVSS 135Anti-PSMA clone 8 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWVRQAPGKGLEWVSDINPAKTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDSYGYRGQGTLVTVSS 136Anti-PSMA clone 9 EVQLVESGGGLVQPGGSLRLSCAASRFMISPYHMHWVRQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCDSYGYRGQGTLVTVSS 137Anti-PSMA clone 10 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWVRQAPGKGLEWVSDINPAGQTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDT AVYYCDSYGYRGQGTLVTVSS 138Anti-PSMA clone 11 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWVRQAPGKGLEWVSDINPAGTTDYAEYVKGRFTISRDNAKNTLYLQMNSLRAEDT AVYYCDSYGYRGQGTLVTVSS 139Anti-PSMA clone 12 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCDSYGYRGQGTQVTVSS 140Anti-PSMA clone 13 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCDSYGYRGQGTQVTVSS

TABLE 10 PSMA Targeting Trispecific Protein Sequences SEQ ID C- NO:Number Construct Sequence 141 C00324 PSMAEVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGK TriTAC CD3GLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKP high aff.EDTAVYYCDGYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTL WYSNRWVFGGGTKLTVLHHHHHH 142C00339 PSMA EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGK TriTAC CD3GLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKP med. aff.EDTAVYYCDGYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKGLEWVARIRSGYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSYTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFNAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCV LWYANRWVFGGGTKLTVLHHHHHH143 C00325 PSMA EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGK TriTAC CD3GLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKP low aff.EDTAVYYCDGYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSLISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSSGAVTSGNYPNWVQQKPGQAPRGLIGGTKFGAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLW YSNRWVFGGGTKLTVLHHHHHH 144C00236 Tool PSMA EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVRQAPGK TriTACGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGGGTKLTVLHHHHHH 145C00362 PSMA p8 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVRQAPGK TriTACGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTL WYSNRWVFGGGTKLTVLHHHHHH 146C00363 PSMA HDS EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGK TriTACGLEWVSDINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKP C363EDTAVYYCDSYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWY SNRWVFGGGTKLTVLHHHHHH 147C00364 PSMA HTS EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGK TriTACGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLKP C364EDTAVYYCDSYGYRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWY SNRWVFGGGTKLTVLHHHHHH 148C00298 PSMA BiTE QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAIISDGGYYTYYSDIIKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARGFPLLRHGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYRYSDVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQYDSYPYTFGGGTKLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY CVLWYSNRWVFGGGTKLTVLHHHHHH149 C00131 EGFR QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGK TriTACEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH 150 C00457 PSMA PH1TQVQLVESGGGVVQAGRSLTLSCAYSGVTVNVYRMGWFRQAPG TriTACKEREFVANINWSGNNRDYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASEKPGRLGEYDYGSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH 151 C00404 PSMA PH1QVQLVESGGGVVQAGRSLRLSCAYSGVTVNVYRMGWFRQAPG TriTACKEREFVANINWSGNNRDYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASEKPGRLGEYDYGSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH 152 C00410 PSMA Z2EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVRQAPGK TriTACGLEWVSTINPAGTTDYAESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWY SNRWVFGGGTKLTVLHHHHHH

TABLE 11 PSMA Binding Domain CDR sequences SEQ ID Nos. SequenceSEQ ID No. 162 RFMISX₁YX₂MH SEQ ID No. 163 X₃INPAX₄X₅TDYAEX₆VKGSEQ ID No. 164 DX₇YGY

TABLE 12 Exemplary Framework Sequences SEQ ID NO: Description Sequence165 Framework (f1) EVQLVESGGGLVQPGGSLTLSCAAS 166 Framework (f2)WVRQAPGKGLEWVS 167 Framework (f3) RFTISRDNAKNTLYLQMNSLRAEDTAVYYC 168Framework (f4) DGYGYRGQGTLVTVSS

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A trispecific protein targeting an epitope within a human prostatespecific membrane antigen (PSMA), wherein said protein comprises (a) afirst domain (A) which is a single chain variable fragment (scFv) thatspecifically binds to a human CD3, comprising a heavy chaincomplementarity determining region (HC CDR) 1, a HC CDR2, a HC CDR3, alight chain complementarity determining region (LC CDR) 1, a LC CDR 2,and a LC CDR 3, wherein the HC CDR1 comprises the amino acid sequence ofSEQ ID NO: 32; the HC CDR2 comprises the amino acid sequence of SEQ IDNO: 42; the HC CDR3 comprises the amino acid sequence of SEQ ID NO: 44,the LC CDR1 comprises the amino acid sequence of SEQ ID NO: 54; the LCCDR2 comprises the amino acid sequence of SEQ ID NO: 79; and the LC CDR3comprises the amino acid sequence of SEQ ID NO: 80; (b) a second domain(B) which is a single domain antibody (sdAb) comprising a CDR1, a CDR2and a CDR3, wherein the CDR1 comprises the amino acid sequence of SEQ IDNO: 100; the CDR2 comprises the amino acid sequence of SEQ ID NO: 104;and the CDR3 comprises the amino acid sequence of SEQ ID NO: 109; and(c) a third domain (C) which is a sdAb that specifically binds to thehuman PSMA, comprising a CDR1, a CDR2, and a CDR3, wherein the CDR1comprises the amino acid sequence of SEQ ID NO: 114; the CDR2 comprisesthe amino acid sequence of SEQ ID NO: 126; and the CDR3 comprises theamino acid sequence of SEQ ID NO: 124; and wherein the human PSMA is a100 kD Type II membrane glycoprotein expressed in prostate tissue. 2.(canceled)
 3. The trispecific protein of claim 1, wherein the firstdomain comprises the amino acid sequence of SEQ ID NO:
 1. 4. The PSMAtargeting trispecific protein of claim 1, wherein the first domain ishumanized or human.
 5. The PSMA targeting trispecific protein of claim1, wherein the first domain has a K_(D) binding of 150 nM or less to CD3on CD3 expressing cells. 6-7. (canceled)
 8. The PSMA targetingtrispecific protein of claim 1, wherein the second domain comprises theamino acid sequence of SEQ ID NO:
 94. 9. The PSMA targeting trispecificprotein of claim 1, wherein the third domain comprises the amino acidsequence of SEQ ID No: 130 or
 140. 10. The PSMA targeting trispecificprotein of claim 1, wherein the domains are linked by linkers L1 and L2,and wherein linkers L1 and L2 are each independently selected from (GS)n(SEQ ID NO: 153), (GGS)n (SEQ ID NO: 154), (GGGS)n (SEQ ID NO: 155),(GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157), or (GGGGS)n (SEQ IDNO: 158), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 11. The PSMAtargeting trispecific protein of claim 10, wherein linkers L1 and L2 areeach independently (GGGGS)₄ (SEQ ID NO: 159) or (GGGGS)₃ (SEQ ID NO:160).
 12. The PSMA targeting trispecific protein of claim 10, whereinthe domains are linked in the order H₂N-(C)-L1-(B)-L2-(A)-COOH.
 13. ThePSMA targeting trispecific protein of claim 1, wherein the protein isless than about 80 kDa.
 14. The PSMA targeting trispecific protein ofclaim 1, wherein the protein is about 50 to about 75 kDa.
 15. The PSMAtargeting trispecific protein of claim 1, wherein the protein is lessthan about 60 kDa.
 16. The PSMA targeting trispecific protein of claim1, wherein the protein has an elimination half-time of at least about 50hours.
 17. The PSMA targeting trispecific protein of claim 1, whereinthe protein has an elimination half-time of at least about 100 hours.18. The PSMA targeting trispecific protein of claim 1, wherein theprotein has increased tissue penetration as compared to an IgG to thesame PSMA.
 19. The PSMA targeting trispecific protein of claim 1,wherein the protein comprises an amino acid sequence of SEQ ID NO: 147or
 152. 20. (canceled)
 21. A pharmaceutical composition comprising (i)the PSMA targeting trispecific protein according to claim 1, and (ii) apharmaceutically acceptable carrier. 22-28. (canceled)
 29. A method oftreating prostate cancer expressing a PSMA, the method comprisingadministration of an effective amount of the pharmaceutical compositionof claim 21 to a subject having said prostate cancer, wherein said humanPSMA is a 100 kD type II membrane glycoprotein expressed in prostatetissue.
 30. (canceled)